Bee venom as an alternative for antibiotics against Staphylococcus aureus infections

The misuse of antibiotics has led to antibiotic-resistant bacterial strains, making it even harder to combat and eliminate their infections. Staphylococcus aureus causes various adverse infections and diseases, including skin abscesses, bloodstream infections, pneumonia, and joint infections. In this study, we aimed to test the cytotoxic and antibacterial effects of bee venom-loaded chitosan nanoparticles (BV-loaded CS-NPs) in comparison to gamma-irradiated BV and native BV from Apis mellifera. The physiochemical characterizations of our treatments were determined by Fourier Transform Infrared Spectroscopy (FTIR), Transmission Electron Microscope (TEM), zeta-potential, release rate, and Encapsulation Efficiency (EE). Our study was conducted on both levels, in-vitro and in-vivo. For the in-vitro study, a bacterial model of Staphylococcus aureus with an ATCC number of 6538 was grown in tryptic soy agar (TSA) medium, and the inhibition zones of our drug candidates were measured with the appropriate statistical analysis performed. For the in-vivo study, levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), Creatinine, Urea, and interleukin 6 (IL-6) were analyzed. BV-loaded CS-NPs showed relatively better results than the other alternatives, which are native BV and gamma-irradiated BV. The results showed that the antibacterial effect of BV-loaded CS-NPs was greater than the alternatives. Furthermore, its cytotoxic effect was far less than the native and irradiated bee venom. These outcomes ensure that loading BV on CS-NPs makes it a promising drug candidate for an antibiotic alternative with minimal cytotoxicity and enhanced antibacterial activity.

www.nature.com/scientificreports/ In-vitro release rate of BV-loaded CS-NPs. 0.1 g of lyophilized BV-loaded CS-NPs was dissolved in 3 ml of phosphate buffer saline (PBS) (0.2 mol/L) at a pH of 7.4. After the preparation of the solution, it was divided into different falcons, each with varying intervals of time (1, 2, 4, 8, 16, 24, 30, and 42 h), and incubated in a water bath (37 °C). Then, the samples were centrifuged at 20,000 × g/9900 rpm (14 °C for 30 min) at determined time intervals. Lastly, the amount of BV released was assessed with Bradford Assay, and BioTek Powerwave XS2 Plate Reader detected the color change 22 .
Bacterial model. The bacteria used was S. aureus with ATCC 6538. It was cultured in TSB for 18 h at 37 °C until the suspension reached OD of 0.5, and the number of bacteria was adjusted to ~ 1.5 × 10 9 CFU/mL at 600 nm 23 .
In-vitro study. The bacteria was inoculated in Tryptic Soya Agar (TSA) media, and its antibacterial effect was tested by the disc diffusion method. The treatments (BV, irradiated BV, CS-NPs, BV-loaded CS-NPs) were used in a concentration of (1 g/L in PBS) the concentrations were adjusted according to the LD 50 of BV that was determined by our previous study 23 and were dissolved in 5 ml PBS, lightly vortexed, discs were soaked in it, then added uniformly to the plate. Ampicillin (10 µg/disc) and Erythromycin (15 µg/disc) discs were used as controls.
The disc diffusion study was repeated 3 times to confirm reproducible data.
In-vivo study. Ethical consideration. All the experimental procedures were performed under the international guidelines for the care and use of laboratory animals. The experimental animal protocol was approved (Protocol Number CUIF5521) by the Institutional Animal Care and Use Committee of Cairo University (CUIA-CUC) (Giza, Egypt). Our study experiments were performed in accordance with ARRIVE guidelines.
Treatments dosage preparation. Mice infection was carried out Intraperitoneally (i.p.) with freshly prepared bacterial suspension ~ 1.5 × 10 8 CFU/mL, and the treatments started 3 h post-infection. All treatments were injected i.p. every 24-h (triple dose). The injected BV dose was 9.02 µg/g mouse weight, according to a previous study 23 . Suspension of BV-loaded CS-NPs was dissolved in PBS (1 mg/ml) and vortexed to prepare an equivalent volume to the BV dosage according to the NPs' Encapsulation Efficiency. Accordingly, similar volumes of BVloaded CS-NPs were used to inject CS-NPs suspension (1 mg/ml). The antibiotic was also injected at the same dose.
Experimental design. Seventy-seven healthy Swiss male albino mice, aged 8-10 weeks (25-35 g), were obtained from the animal house of The Nile Company for Pharmaceuticals & Chemical Industries, Egypt, and given proper housing in terms of diet, water, day/night cycles, and healthy environmental conditions. The animals were divided into 11 groups (7 mice/group), Gp.1: Mice injected with PBS as a control group. Gp.2: Mice injected with BV. Gp.3: Mice injected with gamma-irradiated BV. Gp.4: Mice injected with unloaded CS-NPs. Gp.5: Mice injected with BV-loaded CS-NPs. Gp.6: Mice infected with bacteria. Gp.7: Infected mice treated with BV. Gp.8: Infected mice treated with gamma-irradiated BV. Gp.9: Infected mice treated with unloaded CS-NPs. Gp.10: Infected mice treated with BV-loaded CS-NPs. Gp.11: Infected mice treated with a commercial antibiotic (sulbacef).
Sample collection and biochemical and haematological analysis. All mice were euthanized one-week post-infection with sodium pentobarbital. Blood samples were collected in two different types of tubes, ethylenediaminetetraacetic acid (EDTA) tubes for haematological investigations and serum tubes for biochemical analysis of liver function represented in alanine transaminase (ALT) and aspartate transaminase (AST), Renal function urea and creatinine were measured using (Cat No.: MBS508399), (Cat No.: MBS3805479), and interleukin 6 (IL-6) using (Cat No.: MBS824703), purchased from MyBioSource San Diego (USA), The Mouse IL-6 sandwich ELISA was done according to the protocol of the kit.
Histopathological analysis. After euthanasia, the liver, kidney, thymus, and spleen samples were collected from all experimental groups, fixed in 10% buffered formalin, embedded in paraffin, sectioned into 4-5 µm sections, deparaffinized in xylene, hydrated, and stained with hematoxylin and eosin (H&E), and examined under a light microscope.
Data analysis. The IBM statistical package for the Social Sciences, version 25, was used to analyze the data (copyright by IBM SPSS software, US). For NPs characterization experiments, one sample T-test was applied. For in vitro and in vivo experiments, a one-way Analysis of variance (ANOVA) was used. The smallest level of significance was P < 0.05. Post hoc ANOVA (Tukey's homogeneity test) was used to test the differences and similarities between the experimental groups. All results were represented as a mean ± standard error of the mean (SEM). GraphPad Prism version 8 was used to plot the data (Graph Pad Software Inc., San Diego, CA).

Results
FTIR analysis. The main purpose was to identify the specific peaks for peptides in the BV secondary structure and observe any changes in those peaks in FTIR spectra of irradiated BV. The FTIR spectra of both are shown in (Fig. 1a). In addition to comparing CS-NPs and BV-loaded CS-NPs as shown in Fig. 1b. According to a chemical profiling study, BV showed normal peaks that provide the basic characteristic FTIR spectra of natural venom. The analysis shows significant peaks at approximately 3400 cm −1 and 2940 cm −1 , while the fingerprint region for BV lies between 1700 and 600 cm −1 . This region showed bands with sharp peaks at approximately 1680 cm -1 and 1500 cm −1 , a peak at nearly 1400 cm −1 , and 3 bands at approximately 1050, 945, and 630 cm −1 . All the peaks observed in the spectra of BV are found in the irradiated BV, but they show alteration and have quite lower intensity. The FTIR spectrum of CS-NPs was characterized by three bands (Fig. 1b). A band at 3500-3300 cm −1 related to NH 2 and O-H stretching vibration, a band at 1633 cm -1 refers to NH 2 bending vibration, and a band at 1150 cm −1 corresponds to C-O-C asymmetric stretching vibration. The FTIR spectra of BV-loaded CS-NPs exhibit a distinctive sharp band at 1642 cm -1 , and a peak at 560 cm −1 was observed in both loaded and unloaded NPs.
TEM analysis of NPs. TEM analysis was implemented to study the particle size distribution and morphology of CS-NPs and BV-loaded CS-NPs. The result showed that CS-NPs possess an average size of 24.03 ± 2.22 nm with a smooth surface, while the BV-loaded CS-NPs had an average size of 175.85 ± 23.41 nm, as shown in (Fig. 2).
Zeta potential and PDI. The determined zeta potential and PDI of both CS-NPs and BV-loaded CS-NPs are described in (Fig. 3). A decrease in the zeta potential was observed in BV-loaded CS-NPs, and a significant increase (P < 0.001) in the PDI value was observed in BV-loaded CS-NPs compared to the CS-NPs.

Encapsulation efficiency (EE) of BV-loaded CS-NPs. The optimum concentration of BV and CS for
the preparation of BV-loaded CS-NPs were 550 µg/mL and 2 mg/ml, respectively. The results ensured that by yielding loaded nanoparticles with a high EE of 96.44%.
In-vitro release rate of BV-loaded CS-NPs. As shown in (Fig. 4), the amount of released BV from CS-NPs increased exponentially over time until it reached its optimum release at 30 h. After that specific interval, the amount of BV released began to decrease.
In vitro study. The inhibition zone diameter was measured using the disc diffusion method to compare the antibacterial effect of the treatments against S. aureus, as shown in (Fig. 5a). The treatments gave different inhibition zones, as described in (Fig. 5b).  Biochemical investigations. In this study, the mice group injected with irradiated BV (Gp.3) or BVloaded CS-NPs (Gp.5) showed an increase in ALT, AST, and creatinine levels and a decrease in urea levels compared to the control group (Gp.1). On the other side, the infected groups with S. aureus. Furthermore, infected untreated showed a significant increase (P < 0.01) in ALT levels and a marked increment but not significant in AST, creatinine, and urea levels.
All the treated groups (BV-treated (Gp.7), irradiated BV-treated (Gp.8), CS-NPs treated group (Gp.9), BVloaded CS-NPs treated (Gp.10), and treated group with antibiotic (Gp.11)) showed an increase in all parameters' levels compared to control. Interestingly, the enhancement of BV-loaded CS-NPs on the different parameters was similar to the treated group with the antibiotic (Fig. 7).

Measurement of Interleukin 6 (IL-6).
A major rise in IL-6 levels was spotted, showing that the infection took place. Infected groups treated with BV (Gp.7), irradiated BV (Gp.8), or CS-NPs (Gp.9) showed elevated levels of IL-6 compared to the control group (Gp.1); they had much higher levels of IL-6. The BV-loaded CS-NPs treated group (Gp.10) was the only group to show a decrease in IL-6 levels when compared to the untreated infected group (Gp. 6) or antibiotic-treated group (Gp. 11) (Fig. 8).    www.nature.com/scientificreports/ On the other hand, all of the infected groups except the antibiotic-treated group seemed to have mild congestions and either peri-venular or intra-lobular mild inflammatory infiltrate. Both untreated and CS-NPs treated groups showed mildly dilated central veins, while the BV-loaded CS-NPs treatment showed markedly dilated central vein. Scattered apoptotic hepatocytes only appeared in the irradiated BV-treated sections (Fig. 9).
Histopathological analysis of kidney samples. All kidney sections of non-infected samples showed average glomeruli with average Bowman's spaces as compared to control group (Fig. 10A), with minor changes in the BV group with mildly edematous epithelial lining and markedly congested interstitial blood vessels (Fig. 10B). Both irradiated BV and CS-NPs groups showed mildly congested interstitial blood vessels (Fig. 10C, D). The changes increased in the BV-loaded CS-NPs group with scattered apoptotic epithelial lining and markedly dilated congested blood vessels (Fig. 10E).
The infected groups with S. aureus showed different levels of kidney damage due to bacterial infection. The damage in the CS-NPs group was the highest, showing scattered small-sized glomeruli with narrow Bowman's spaces, scattered apoptotic epithelial lining, and mildly congested blood vessels (Fig. 10D*). The damage decreased in the irradiated BV group showing widened Bowman's spaces and scattered apoptotic epithelial lining (Fig. 10C*). The BV and BV-loaded CS-NPs groups showed average conditions similar to control and antibiotic groups with a minor change in the interstitial blood vessels, which were mildly congested ( Fig. 10B*, E*, F*). These groups were compared to the infected group with S. aureus (Fig. 10A*).
Histopathological analysis of spleen samples. All non-infected groups showed average lymphoid follicles, but there was mild congestion in blood vessels of the BV group along with scattered apoptosis and similar congestion in the blood sinusoids of the CS-NPs group. Excess giant cells were also observed in the irradiated BV and CS-NPs groups. These observations leave the BV-loaded CS-NPs group the most similar to the control with both average blood sinusoids and scattered giant cells.
For the infected groups, the spleen sections of the no-treatment group showed markedly congested blood sinusoids, scattered apoptotic cells, and excess giant cells. The congestion decreased in the gamma-irradiated BV and CS-NPs groups, while it completely disappeared in the BV, BV-loaded CS-NPs, and antibiotic-treated groups. All groups showed average-sized lymphoid follicles except for the gamma-irradiated BV, and the presence of excess giant cells was also observed in all groups except for the gamma-irradiated BV and CS-NPs groups (Fig. 11).
Histopathological analysis of thymus samples. All of the non-infected groups showed thymus sections similar to the control but with some minor variations, as the BV group showed mild congestion in blood vessels, while both the CS-NPs and BV-loaded CS-NPs showed scattered apoptotic thymocytes in the cortex.
Meanwhile, there were great variations in the thymus sections of infected groups. The untreated group showed a detached capsule, cortex with markedly apoptotic thymocytes, and average reticular epithelial cells. Excess reticular cells in the medulla were observed in all other groups except BV-loaded CS-NPs. BV, gamma-irradiated BV, and antibiotic-treated groups also showed cortex with scattered apoptotic thymocytes. The BV-loaded CS-NPs www.nature.com/scientificreports/ treated group sections were very similar to the control with average cortex, average thymocytes, and average medulla with average reticular cells (Fig. 12).

Discussion
The number of ARB strains increases annually, rendering most antibiotics ineffective, leading to it being one of the largest causes of deathaused by continuous antibiotic abuse 4 . Honeybee venom extracted from A. mellifera can potentially be a therapeutic agent for different diseases. Nevertheless, it is a toxin that can lead to several consequences 24 . So, our goal is to study its therapeutic effect by testing the antibacterial activity while eliminating some of its cytotoxicity by treating BV with gamma radiation and loading the BV on CS-NPs. The CS-NPs were prepared by the ionic gelation method, which depends mainly on electrostatic interactions. TEM has established the hypothesis of successfully incorporating BV on CS-NPs due to the size variation between the BV-loaded CS-NPs and unloaded CS-NPs. This was further confirmed by the results of the release rate and EE showing an excellent BV uptake by the CS-NPs. Furthermore, the surface charge of NPs decreased upon loading of BV, which was later confirmed by the FTIR results. All of these results were in accordance with previous studies 25,26 .
BV is composed of a complex of proteins that several methods could characterize. Exposing BV to gamma radiation leads to changes in the conformation of those components, which leads to the molecular weight change of BV. So, FTIR analysis was performed for further confirmation of different forms of BV. The comparative FTIR analysis of BV and irradiated BV proved the purity of obtained venoms, as there was no presence of any bands other than those of the venom itself. In addition, all of the peaks shown in the spectra of BV were relevant to its actual composition. Alteration of those bands in the spectra of gamma-irradiated BV proved the change in the functional groups caused by radiation. The impact of those changes is later analyzed by comparing the antibacterial activity and cytotoxic effect of both BV and irradiated BV 27 .
The comparative FTIR spectra of CS-NPs and BV-loaded CS-NPs also confirmed the uptake of BV by NPs, showing the specific bands for nanoparticles in both the unloaded BV-loaded CS-NPs spectra, in addition to the sharp band for BV at 1642 cm −1 indicating that BV molecules were absorbed on the NPs' surface. Phosphoric ions www.nature.com/scientificreports/ were also present. Consequently, the NPs FTIR spectra were compatible with the result of a phosphate-modified CS film. The phosphate groups of Na-TPP were attached to amino groups of CS molecules in NPs. TPP served as a cross-linker during preparation, forming additional hydrogen bonds with amino groups on CS and BV. The in vitro disc diffusion method tested the difference between the antibacterial activity of irradiated BV and BV-loaded CS-NPs. It showed that the loaded NPs have more antibacterial activity than irradiated BV according to their respective diameters of the inhibition zones.
Hematological investigations showed no significant results in different mice groups compared to the control group; however, they show a relevant decrease in WBCs, which indicates the infection and liver damage in infected groups 28 , in non-infected groups, WBCs decreased due to the cytotoxicity of BV and CS as an immune response against CS-NPs 29 . Also, treated infected groups, especially those treated with CS-NPs, with BV-loaded CS-NPs or with antibiotics, showed results similar to the normal range, which means they have suitable antibacterial activity with low cytotoxicity.
In the non-infected groups, the liver functions showed a decrease in ALT levels of BV-treated mice, indicating the anti-inflammatory effect of BV 30 and an increase in AST levels due to the presence of PLA2, which leads to liver injury 31 . These results were consistent with the liver sections, which showed mildly vacuolated cytoplasm as evidence of liver injury and are consistent with previous studies 32 . The increase in ALT and AST levels in the irradiated BV-treated group was due to the alteration in the protein content structure of BV due to radiation which affected its anti-inflammatory effect. The increased levels of ALT and AST in CS-NPs and BV-loaded CS-NPs treated groups indicate the protective role of the NPs on the hepatocytes 33 and are also supported by average hepatocytes in liver sections. After the bacterial infection, ALT and AST levels increased in all infected treated groups compared to the control group. Histopathological sections of liver results also showed a mild inflammatory infiltrate in all infected treated groups, leading to fibrogenesis 34 .
The kidney is the second organ exposed to infection. Kidney sections of the control group showed average glomeruli with average Bowman's spaces, average proximal tubules with preserved brush borders, average distal tubules, and average interstitial blood vessels. These histopathological results started to change in the BVinjected group showing mildly edematous epithelial lining and markedly congested interstitial blood vessels, leading to increased renal interstitial pressure affecting the entire capillary bed and tubules possibly causing local hypoxia 35 . The inflammatory response decreased in irradiated BV and CS-NPs injected groups showing mildly congested interstitial blood vessels. Meanwhile, the BV-loaded CS-NPs injected group showed a huge change in its kidney sections with scattered apoptotic epithelial lining and markedly dilated congested blood vessels. These histopathological results of the kidney were supported by kidney function results showing a slight increase in creatinine levels and a decrease in urea levels.
In the case of infected groups with S. aureus, the treatment with irradiated BV showed atrophied glomeruli with widened spaces and scattered apoptotic epithelial lining. The irradiated BV-treated group showed a reduction in urea levels and a high increase in creatinine levels as an indicator of severe bacterial infection with a high cytotoxic effect of irradiated BV 36,37 . The cytotoxicity began to decrease in the BV-treated group, indicating a decrease in urea and creatinine levels, while increasing levels of creatinine and decreasing urea levels in the Spleen and thymus are two of the most important immune-regulatory organs 38,39 ; previous studies proved that BV is a double-edged sword and could either trigger or suppress inflammatory and immune responses, the optimum treatment of BV would have an antibacterial effect without causing an allergic reaction 40 . Analysis of histological sections for the groups relative to their corresponding IL-6 levels showed that the control group had average spleen and thymus tissues. IL-6 levels of the non-infected groups were all slightly similar to the control with the injection and the BV-loaded CS-NPs groups being the closest to the control and the irradiated BV-treated group the most deviated, and this was further shown in the histology samples with the presence of excess giant cells in the spleen sections of irradiated BV injected group and a less amount of them in the BVloaded CS-NPs. These giant cells result from numerous macrophage fusions due to high inflammation 41 . It was also notable that the CS-NPs might have induced an inflammatory response as it resulted in mild congestion and the presence of excess giant cells in the spleen and cortical apoptosis in the thymus, which is usually a result of infections or cytotoxicity 42 , we suggest that this could be due to the presence of impurities as CS-NPs normally play anti-inflammatory roles 43 .
Moreover, interleukin-6 (IL-6) which is a multifunctional cytokine that plays a crucial role in host defense due to its vast array of immunological and hematopoietic activities and its robust capacity to activate the acute  42,44 , only seemed to decrease in the case of the BV-loaded CS-NPs treated group, which had average spleen and thymus sections and an IL-6 level that was very close to the negative control. All of the other drugs caused a significant increase in IL-6 levels in the infected groups. The irradiated BV also showed the most significant increase treated group, whose spleen sections along with the CS-NPs treated group sections, had mild congestions and excess giant cells. Thymus sections of the irradiated BV group also showed cortical apoptosis and increased medullary reticular cells. The overall summary of results suggested that, unlike radiation, loading the BV on CS-NPs gave it the most efficient antibacterial activity without triggering an inflammatory response or causing major damage to the thymus or spleen.
In conclusion, BV-loaded CS-NPs have shown relatively better results than the other alternatives, which are native BV and irradiated BV. The In-vitro study showed that the antibacterial effect of BV-loaded CS-NPs was greater than the alternatives. Furthermore, its cytotoxic effect was far less than the native and irradiated BV. These outcomes reinforce that BV-loaded CS-NPs may indeed be an adequate alternative to antibiotics and could replace them with some dedicated search.

Data availability
The datasets generated and/or analysed during the current study are not publicly available due but are available from the corresponding author on reasonable request.